The discovery of the first imine reductases with a broad substrate scope resulted
in identification of a broad range of additional enzymes via a sequence homology
search. The hits have been used to generate an electronic library of several
hundred putative imine reductases [1059]. Most imine reductases known to date
originate from Streptomyces, but are also found in Mycobacteria, Bacillus
and Pseudonocardia sp., and their physiological role and hence their natural
substrate(s) are unknown. Although positively charged N-alkylated iminium species are reduced, electronically related carbonyl compounds are unreactive
[1060]. Mechanistically, imine reductases do not possess a metal ion (such as
Zn
2+ ) found in many alcohol dehydrogenases, but act through general acid-base
catalysis. Imine reduction is assumed to proceed through hydride delivery from
NAD(P)H onto the electrophilic imine carbon with concomitant protonation at
N involving an Asp, His or Tyr residue acting as Brønsted acid to overcome the
formation of an (energetically unfavourable) amide intermediate (R 2 N
À
). The large
majority of imine reductases prefers NADPH as cofactor, only a few enzymes can
use both nicotinamide species about equally well.
Reductive Amination of Ketones
In contrast to the reduction of hydrolytically stable cyclic imines which constitutes
a viable protocol for the formation of cyclic sec-amines, open-chain imines derived
from ketones and ammonia or short-chain prim-amines (e.g. methyl- or nbutylamine) are converted by imine reductases at low rates, which results in low
to modest conversions (typically 50–70%) and requires high enzyme loadings
[1061, 1062]. In addition, whole-cell preparations and crude cell lysates containing
imine reductases are plagued with competing ketone reduction by alcohol dehydrogenases and the amine donor has to be employed in excess (typically ~50:1) to
drive the equilibrium towards imine formation. Recently, imine reductases were
identified which catalysed the reductive amination of cyclic, aliphatic and aromatic
ketones (e.g. 2-hexanone or cyclohexanone) using ammonia and small aliphatic
prim-amines (preferably methylamine) with encouraging results [1063].
Reductive Amination of α-Ketocarboxylic Acids
The (reversible) transformation of an α-ketocarboxylic acid in presence of ammonia and one equivalent of NAD(P)H furnishes the corresponding α-amino acid and
is catalyzed by amino acid dehydrogenases [EC 1.4.1.X] [1064]. This reaction bears
a strong resemblance to imine reduction and it formally represents a reductive
amination (Scheme 2.131). A vast number of L-amino acid dehydrogenases from
diverse organisms as well as variants engineered for industrial application has been
described [1065–1068]. Stereo-complementary D-selective enzymes have been
developed via protein engineering of meso-diaminopimelic acid D-dehydrogenases
[1069, 1070] and they have been applied to the pilot-plant scale production of the
non-natural amino acid D-5,5,5-trifluoromethylnorvaline [1071].
As deduced for L-Leu-dehydrogenase [1072], the α-ketoacid substrate is positioned in the active site between two Lys-residues (Scheme 2.131). Nucleophilic
attack by NH 3 leads to a hemiaminal intermediate, which eliminates H 2 O to form an
iminium species. The latter is reduced by a hydride from nicotinamide forming the
2.2 Reduction Reactions
159
in identification of a broad range of additional enzymes via a sequence homology
search. The hits have been used to generate an electronic library of several
hundred putative imine reductases [1059]. Most imine reductases known to date
originate from Streptomyces, but are also found in Mycobacteria, Bacillus
and Pseudonocardia sp., and their physiological role and hence their natural
substrate(s) are unknown. Although positively charged N-alkylated iminium species are reduced, electronically related carbonyl compounds are unreactive
[1060]. Mechanistically, imine reductases do not possess a metal ion (such as
Zn
2+ ) found in many alcohol dehydrogenases, but act through general acid-base
catalysis. Imine reduction is assumed to proceed through hydride delivery from
NAD(P)H onto the electrophilic imine carbon with concomitant protonation at
N involving an Asp, His or Tyr residue acting as Brønsted acid to overcome the
formation of an (energetically unfavourable) amide intermediate (R 2 N
À
). The large
majority of imine reductases prefers NADPH as cofactor, only a few enzymes can
use both nicotinamide species about equally well.
Reductive Amination of Ketones
In contrast to the reduction of hydrolytically stable cyclic imines which constitutes
a viable protocol for the formation of cyclic sec-amines, open-chain imines derived
from ketones and ammonia or short-chain prim-amines (e.g. methyl- or nbutylamine) are converted by imine reductases at low rates, which results in low
to modest conversions (typically 50–70%) and requires high enzyme loadings
[1061, 1062]. In addition, whole-cell preparations and crude cell lysates containing
imine reductases are plagued with competing ketone reduction by alcohol dehydrogenases and the amine donor has to be employed in excess (typically ~50:1) to
drive the equilibrium towards imine formation. Recently, imine reductases were
identified which catalysed the reductive amination of cyclic, aliphatic and aromatic
ketones (e.g. 2-hexanone or cyclohexanone) using ammonia and small aliphatic
prim-amines (preferably methylamine) with encouraging results [1063].
Reductive Amination of α-Ketocarboxylic Acids
The (reversible) transformation of an α-ketocarboxylic acid in presence of ammonia and one equivalent of NAD(P)H furnishes the corresponding α-amino acid and
is catalyzed by amino acid dehydrogenases [EC 1.4.1.X] [1064]. This reaction bears
a strong resemblance to imine reduction and it formally represents a reductive
amination (Scheme 2.131). A vast number of L-amino acid dehydrogenases from
diverse organisms as well as variants engineered for industrial application has been
described [1065–1068]. Stereo-complementary D-selective enzymes have been
developed via protein engineering of meso-diaminopimelic acid D-dehydrogenases
[1069, 1070] and they have been applied to the pilot-plant scale production of the
non-natural amino acid D-5,5,5-trifluoromethylnorvaline [1071].
As deduced for L-Leu-dehydrogenase [1072], the α-ketoacid substrate is positioned in the active site between two Lys-residues (Scheme 2.131). Nucleophilic
attack by NH 3 leads to a hemiaminal intermediate, which eliminates H 2 O to form an
iminium species. The latter is reduced by a hydride from nicotinamide forming the
2.2 Reduction Reactions
159